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Isolation and Molecular Characterization of Mycoplasma Spp Veterinary World, EISSN: 2231-0916 RESEARCH ARTICLE Available at www.veterinaryworld.org/Vol.12/May-2019/6.pdf Open Access Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt Mounier M. Abdel Halium1, Fayez A. Salib1, S. A. Marouf2 and Emil S. Abdel Massieh1 1. Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt; 2. Department of Microbiology and Mycology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt. Corresponding author: Emil S. Abdel Massieh, e-mail: [email protected] Co-authors: MMAH: [email protected], FAS: [email protected], SAM: [email protected] Received: 04-12-2018, Accepted: 15-03-2019, Published online: 13-05-2019 doi: 10.14202/vetworld.2019.664-670 How to cite this article: Abdel Halium MM, Salib FA, Marouf SA, Abdel Massieh ES (2019) Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt, Veterinary World, 12(5): 664-670. Abstract Background and Aim: Different species of Mycoplasma are associated with many pathological problems in small ruminants including respiratory manifestation, this problem results in significant losses, especially in African countries. This study aimed to (I) study some epidemiological aspects of Mycoplasma species infections in Egyptian sheep and goats at Giza Governorate, (II) diagnosis of Mycoplasma species affections using bacterial isolation and identification, (III) apply the polymerase chain reaction (PCR) for typing of different Mycoplasma species, and (IV) illustrate the phylogenetic tree for the isolated Mycoplasma species and other species from GenBank using the purified PCR product. Materials and Methods: A total of 335 samples were collected from sheep and goats from Giza Governorate in Egypt as 142 nasal swabs from clinically affected animals, 167 pneumonic lungs, 18 samples from tracheal bifurcation, and 8 samples by bronchial wash were cultured on pleuropneumonia-like organisms (PPLOs) media for cultivation of Mycoplasma species. PCR and sequencing and phylogenetic analysis were adopted to identify and classify the isolated Mycoplasma species. Results: A total of 24 Mycoplasma isolates were isolated on PPLO media, identified by biochemical tests, and confirmed and typed by PCR using specific primers. 10 isolates were confirmed as Mycoplasma arginini, four isolates as Mycoplasma ovipneumoniae by PCR, and 10 isolates as undifferentiated Mycoplasma species. A purified isolate of M. arginini and M. ovipneumoniae was sequenced and phylogenetic analysis was illustrated. Conclusion: M. arginini and M. ovipneumoniae are prevalent in Egyptian sheep and goats. Further studies on M. arginini are required due to its high frequency of isolation from pneumonic sheep and goats and also from animals suffer from different respiratory manifestations. Keywords: goats, Mycoplasma, polymerase chain reaction, sequencing and phylogenetic analysis, sheep. Introduction and mastitis [5]. Mycoplasma species commonly Mycoplasma belongs to a group of bacteria associated with pneumonia in small ruminants are named Mollicutes which characterized by its min- Mycoplasma ovipneumoniae, Mycoplasma arginini, ute genome size and perpetually devoid of the cell Mycoplasma capri, Mycoplasma capripneumoniae, wall [1]. Different Mycoplasma species are accompa- and Mycoplasma capricolum [6]. M. arginini is fre- nying by many diseases and problems in both mam- quently isolated with M. ovipneumoniae from cases of malian and avian species [2], sheep and goats (poor atypical pneumonia in sheep and goats [5] and other man’s cow) are economically critical in many coun- cases with lung consolidation [7]. M. arginini is also tries including Egypt as consumed for meat, wool, and isolated from other locations such as genital organs, milk production, more than 10% of meat production eyes, and ears [8]; in addition, isolation of M. arginini in Egypt is from sheep and goats [3]. from pathological cases in human [9,10] gives suspi- Mycoplasma infections leading to a great eco- cion that M. arginini may have zoonotic importance. nomic loss due to it cause high morbidity and mor- In Egypt, different Mycoplasma species have been tality rates in sheep and goats populations in African isolated including M. arginini, M. ovipneumoniae, countries including Egypt, European countries, and and Mycoplasma agalactiae [11]. India [4]. Mycoplasma causes various clinical man- The respiratory problems are considered as mul- ifestations as pneumonia, conjunctivitis, arthritis, tifactorial disease where interaction between different microbial agents such as bacteria (Pasteurella and Copyright: Abdel Halium, et al. Open Access. This article is Mycoplasma), viruses (PI3, reovirus, and adenovi- distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ rus), and fungi and also other factors as host defense by/4.0/), which permits unrestricted use, distribution, and mechanism and environmental factors including cli- reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the matic conditions and stress of transportations lead to Creative Commons license, and indicate if changes were made. increase the incidence of those problems [12]. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data Mycoplasma is highly fastidious microorganism made available in this article, unless otherwise stated. required very precise media to develop in vitro, the Veterinary World, EISSN: 2231-0916 664 Available at www.veterinaryworld.org/Vol.12/May-2019/6.pdf low ability of Mycoplasma to form macromolecules Postmortem examination needed for their growth refers to their evolutionary Lung tissues, tracheas, and tracheal bifurcation development from other bacteria; it is highly sus- of slaughtered sheep and goats were examined to pected that many Mycoplasmas exist in nature, how- detect and record different pathological changes [15]. ever, have not been isolated due to their hard growth Epidemiological investigation in vitro on artificial media [1]. The prevalence of isolation of different Lately, the high development of polymerase Mycoplasma species in the examined sheep and goats chain reaction (PCR) technique makes the detection was recorded according to species, ages, sexes, and of different species of Mycoplasma using specific season of the year [16]. primers much simpler, till now, PCR remains the most valuable and rapid method for detecting specific spe- Bacterial isolation and identification of Mycoplasma species cies of Mycoplasma [13]. Moreover, the amplified For nasal swab from living sheep and goats with purified PCR products of many genes from differ- respiratory manifestation, sterile cotton swabs were ent strains can be sequenced, offering the chance for used to collect nasal swabs from nostrils of affected whole or partial typing of various species and strains at a more accurate level allowing molecular epidemi- sheep and goats, and they were taken on pleuro- ological studies and research. pneumonia-like organisms (PPLOs) broth placed in The molecular epidemiological analysis allows an icebox and submitted directly to the laboratory. the genotyping of different strains and has used in the Serial dilutions from nasal swab were formed and tracing, control, and prevention of some diseases; also, incubated on PPLO broth of pH 7.4-7.6 with 15% molecular typing of some strains such as M. arginini heat-inactivated swine serum, 10% fresh yeast extract, would help in strain differentiation and then know the and 0.0005 g/ml thallium acetate at 37°C for 3-7 days, suspected pathological importance of these strains [8]. then cultured on solid PPLO media by running drop This study aimed to (I) study some epidemiological technique [17]. aspects of Mycoplasma species infections in Egyptian For bronchial washes, approximately 1 cm long sheep and goats at Giza Governorate, (II) diagnosis of skin incision was done with a surgical scalpel over the Mycoplasma species affections using bacterial isola- midpoint of the trachea under local anesthesia of 2% tion and identification, (III) apply the PCR for typing lidocaine with epinephrine, a 16-gauge hypodermic of different Mycoplasma species, and (IV) illustrate needle was inserted into the tracheal lumen. A catheter the phylogenetic tree for the isolated Mycoplasma equipped with two-way lumen connect valve was then species and other species from GenBank using the inserted through the needle up to bronchial bifurca- purified PCR product. tion; approximately 20-50 ml of Hank’s balanced salt solution was injected through the catheter and then Materials and Methods immediately aspirated with syringe. The debris was Ethical approval then removed by low-speed centrifugation; and then, Animal ethical approval was obtained from the samples were cultured on PPLO broth for 3-7 days Institutional Animal Care and Use Committee, Cairo at 37°C and then cultured on solid PPLO by running University (II F 23 18). drop technique [18,19]. Animals and samples For lung tissues and tracheal bifurcations from A total of 246 sheep and 89 goats of different ages, slaughtered sheep and goats, 167 samples from pneu- sexes, and breeds suffering from respiratory manifes- monic areas and 18 samples from tracheal bifurcation tations were examined clinically and bacteriology for were aseptically
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